In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of fluorescent-derivatives of our tropane based DAT inhibitors. Our fluorescent tropane-based ligand, JHC1-064, has been used in many labs to characterize the trafficking and cellular distribution of SERT and DAT in living neuronal cells. Indeed, recent experiments in living neurons with JHC 1-064 have provided data that challenge mechanistic dogma for transporter translocation, as determined in DAT transfected heterologous cells, which is one area of ongoing research with these agents. This fluorescent ligand has been used in a clinical study to confirm the loss of DAT at the cell membrane in a patient with a genetically mutated DAT that was related to his Parkinsonian and attention deficit comorbidities. Recently, in order to examine the mechanisms of DAT filopodial targeting, we used quantitative live-cell fluorescence microscopy to compare the effects of the DAT inhibitor cocaine and its fluorescent analog JHC1-64 on the plasma membrane distribution of wild-type DAT and two non-functional mutants, R60A and W63A that are not accumulated in filopodia. W63A did not bind JHC1-64, whereas R60A bound this compound, although less efficiently than did wild-type DAT. Molecular dynamics simulations predicted that R60A preserves the outward-facing conformer to some degree through compensatory intracellular salt bridging and is therefore capable of cocaine binding. Imaging analysis showed that JHC1-64 occupied R60A mutant was preferentially concentrated in filopodia, whereas free R60A mutant DAT protein was evenly distributed within the plasma membrane. Cocaine binding significantly increased density of R60A but not W63A in filopodia. Further, zinc binding, known to stabilize the OF state, also increased R60A concentration in filopodia. Finally, amphetamine, that is thought to disrupt DAT OF conformation, reduced the concentration of wild type DAT in filopodia. Altogether, these data indicate that the outward facing conformation of DAT is required and sufficient for its effective targeting to and accumulation in filopodia. Further, JHC 1-064 binds with high affinity to the serotonin transporter (SERT) and the norepinephrine transporter (NET), as well, and we are conducting analogous studies, first in cell lines to study trafficking and cellular distribution of these other monoamine transporters. This project has led to the design of novel fluorescent ligands, using customized fluorophores suitable for live super resolution imaging. Knowledge of the monoamine transporters structural elements that define substrates and inhibitors is sparse. We have recently addressed this structure-activity question directly by generating a series of 3,4-methylenedioxy ring-substituted amphetamine analogs that differ only in the number of methyl substituents on the amine group. Starting with 3,4,-methylenedioxy-N-methylamphetamine (MDMA), 3,4,-methylenedioxy-N,N-dimethylamphetamine (MDDMA) and 3,4,-methylenedioxy-N,N,N-trimethylamphetamine (MDTMA) were prepared. We evaluated functional activities of the analogues at all three monoamine transporters in native brain tissue and in cells expressing transporters, and used ligand docking to generate models of the respective protein-ligand complexes. This approach allowed us to relate experimental findings to available structural information. Our results suggest that 3,4-methylenedioxy amphetamine analogs bind at the orthosteric binding site (OBS) of transporters by adopting one of two mutually exclusive binding modes. MDA and MDMA adopt a high-affinity binding mode, whereas MDDMA and MDTMA adopt a low-affinity binding mode in which the ligand orientation is inverted. Importantly, MDDMA can alternate between both binding modes while MDTMA exclusively binds to the low affinity mode. Our experimental results are consistent with the idea that the initial orientation of bound ligands is critical for subsequent interactions that lead to transporter conformational changes and substrate translocation. In a separate project, we have prepared photoaffinity ligands, based on DAT or SERT inhibitors in order to identify their binding sites at the transporters. Our previously reported crosslinkable photoactive cocaine analog, N-4-(4-azido-3-I-iodophenyl)-butyl-2-carbomethoxy-3-(4-chlorophenyl) tropane, MFZ 2-24 adducts to a fragment of TM1 of DAT between residues 67 and 80. We recently used complementary computational, biochemical docking, and substituted cysteine accessibility strategies to identify the single amino acid adduction site and binding pose of MFZ 2-24 on DAT. Our findings reveal that MFZ 2-24 occupies the central binding pocket (S1 pocket), with the azide group on MFZ 2-24 adducting to Leu80 in TM1 and the tropane ring nitrogen coordinating with the negatively charged carboxyl side chain of Asp79. The placement and orientation of the MFZ 2-24 tropane pharmacophore is consistent with that of the cocaine-like photoaffinity ligand RTI 82 which crosslinks to TM6. The superimposition of the tropane ring in binding poses of these two ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and a competitive mechanism of DA uptake inhibition. These methodologies provide a basis for elucidating the binding site and orientation of atypical DAT inhibitors such as the benztropines (e.g., JHW007) to understand the paradoxical outcome of DAT blockade by these compounds. We have now expanded this work to include a series of deconstructed ibogaine analogues. Nor-ibogaine has recently been described as a pharmacochaperone, capable of rescuing mutant DATs that are unable to fold properly in the endoplasmic reticulum, so never make it to the membrane, leaving the patient with poor DAT function that results in movement and neuropsychiatric disorders. Hence we have embarked on a screening and synthesis project to identify lead molecules that may rescue these mutant DATs and provide the opportunity to improve DAT function and ameliorate symptoms associated with these disorders.